Computational protocol: Traumatic Brain Injury Upregulates Phosphodiesterase Expression in the Hippocampus

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Protocol publication

[…] At 24 h after sham surgery (n = 6) or TBI (n = 6), animals were deeply anesthetized (3% isoflurane, 70% N2O, 30% O2, 5 min) and transcardially perfused with PBS (120 mL, 4°C). The ipsilateral hippocampus was dissected at 4°C. Tissue was mechanically dissociated into single cell suspension and cells were labeled with CD45 Alexa 647 (202212, 1.25 μg/ml, BioLegend) and CD11b v450 (53-4321-80, 1 μg/ml, eBioscience). Dead cells were excluded using LIVE/DEAD Fixable Near-IR dead cell stain (L10119, 1 μl/ml, Life Technologies). Cells were fixed and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization kit (554714, BD Biosciences). Cells were intracellularly labeled with phospho-PDE4A (GTX14610, 2 μg/ml, GeneTex), PDE4B2 (ABS181, 2 μg/ml, EMD Millipore) or PDE4D (sc-25814, 2 μg/ml, Santa Cruz Biotechnology). PDE staining was detected with PE-conjugated secondary antibodies (12-4739-81, 10 μg/ml, eBioscience). Flow cytometry data was acquired on a BD LSR II flow cytometer with four emission lasers at 407, 488, 532, and 640 nm. Data collection was performed using BD FACSDiva 8.0.1 (BD Biosciences) and analyzed with Kaluza 1.2 software (Beckman Coulter). […]

Pipeline specifications

Software tools BD FACSDiva, Kaluza
Application Flow cytometry
Organisms Rattus norvegicus
Diseases Brain Injuries, Cerebrovascular Trauma
Chemicals Rolipram